Increased leukocyte trafficking and infiltration of the vasc
Increased leukocyte trafficking and infiltration of the vascular wall are thought to provide a key stimulus for SMC activation and subsequent neointima formation in response to vascular injury , . The essential trigger regulating the accumulation of leukocytes in the vessel wall is the upregulation of cell adhesion molecules including selectins, ICAM-1 and VCAM-1 , . As the degree of the inflammatory response is directly correlated with the extent of subsequent neointimal thickness, inhibition of cell adhesion molecules such as VCAM-1 has been demonstrated to reduce neointimal mass , . The regulation of cell adhesion molecule expression and leukocyte trafficking has repeatedly been linked to cAMP-Signaling. CAMP-elevating agents have previously been found to inhibit TNF-α mediated induction of genes that regulate leukocyte adhesion such as selectins, ICAM-1 and VCAM-1 , . Furthermore, selective PDE4 inhibition by roflumilast impedes leukocyte-endothelial cell interactions and expression of P- and E-selectin in endothelial Vilazodone Hydrochloride sale in vitro  and reduces leukocyte trafficking from lung microvasculature into airways and lung parenchyma in in vivo models of chronic obstructive pulmonary disease , . While roflumilast has been found to be significantly more potent in inhibiting leukocyte endothelial cell interactions than the structurally unrelated PDE4 inhibitors rolipram or cilomilast , its effect on cell adhesion molecule expression in SMCs has not been evaluated so far. In this study we could demonstrate that roflumilast treatment significantly reduced VCAM-1 expression in SMCs, while we could only observe limited effects on other cell adhesion molecules. This inhibition of VCAM-1 expression was functionally also associated with a reduction of monocyte adhesion in vitro. Although several investigators have previously sought to elucidate the mechanisms of cAMP-mediated regulation of adhesion molecule expression, there have been inconclusive results , , . This could be explained by the high level of sophistication of cAMP-signaling as well as the fact, that most of these studies were performed prior to the discovery of Epac as a second effector molecule of cAMP-signaling. Here we report for the first time to our knowledge that inhibition of TNF-α mediated VCAM-1 expression in SMCs is conferred through Epac rather than PKA activation. Using the recently discovered Epac- and PKA-specific agonists 8CPT-2-O-Me-cAMP and 6-MB-cAMP , , inhibition of VCAM-1 expression was observed following Epac activation, while specific PKA activation had no effect. Furthermore, inhibition of Epac with the new specific Epac inhibitor ESI-09  as well as siRNA-mediated knock-down completely blocked inhibition of VCAM-1 expression by roflumilast, confirming the role of Epac-dependent signaling in VCAM-1 regulation. In our study roflumilast treatment strongly inhibited VCAM-1 expression with minor effects on the expression of other cell adhesion molecules. A similar phenotype has been described in endothelial cells and linked to epigenetic posttranslational histone modifications of the VCAM-1 promoter , . Furthermore, roflumilast treatment had no effect on NF-κB translocation as well as VCAM-1 promoter activity, suggesting the involvement of regulatory components beyond the repression of transcription factor-dependent transactivation. As histone modifications control the accessibility of a transcription factor to a gene, they constitute a hierarchic upper-level of transcriptional control regulating gene expression in a specific manner . Particularly methylation of H3K4 has been described as an activating histone mark that is highly enriched at the transcriptional start site (TSS) of active genes and induced following inflammatory stimulation , . Therefore, we analyzed histone H3 lysine 4 dimethylation (H3K4me2) at the TSS of the VCAM-1 promoter. Correspondingly, we found an induction of H3K4me2 at the VCAM-1 promoter in SMCs following TNF-α stimulation. This induction was significantly attenuated by treatment with roflumilast as well as Epac activation, while PKA activation had no effect on H3K4me2 at the VCAM-1 promotor region. Interestingly, Epac has previously been shown to interact with histone deacetylases (HDACs) a class of enzymes known to control gene activation by regulating histone acetylation , . Furthermore, HDACs are also known to govern the methylation and demethylation of H3K4 in a wide variety of inducible genes , , and HDAC inhibition, particularly inhibition of class I HDACs, leads to increased methylation at the respective gene promoters , . In line with these observations, we found that treatment with the specific class I HDAC inhibitor MGCD0103 was sufficient to completely block the inhibitory effect of roflumilast on VCAM-1 expression. Together, these data provide a possible mechanism for PDE4-dependent regulation of VCAM-1 expression in SMCs through Epac-dependent epigenetic mechanisms (Fig. 6).